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First patch, then catch: measuring the activity and the mRNA transcripts of a proton pump in individual Lilium pollen
Renate Gehwolf1, Martin Griessner, Heidi Pertl
1Institut für Pflanzenphysiologie, University of Salzburg, Hellbrunnerstr. 34, 5020, Salzburg, Austria.
Abstract:
Combining the patch-clamp method with single-cell reverse transcription polymerase chain reaction (scRT-PCR) a fusicoccin-induced current reflecting the activity of the plasma membrane H(+) ATPase of lily pollen protoplasts was measured and subsequently, the ATPase-encoding mRNAs were collected and amplified. Southern blot signals were observed in all 'patch-catch' experiments and could be detected even in 2560-fold dilutions of the pollen contents. H(+) ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation. In 15% of the scRT-PCR experiments, a random non-reproducibility of the PCR was observed, probably caused by varying amounts of ATPase mRNAs in the protoplasts.